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Updated: Jun 29, 2025
Visualization, Quantification, and Mapping of Immune Cell Populations in the Tumor Microenvironment
Published on: March 25, 2020
Imaging Mass Cytometry for In Situ Immune Profiling
Kevin Hu1,2, Andrew Harman1,2, Heeva Baharlou3,4
1Centre for Virus Research, The Westmead Institute for Medical Research, Westmead, NSW, Australia.
Insights
This study presents an optimized protocol for imaging mass cytometry (IMC) combined with RNAscope, enabling simultaneous protein and RNA detection in tissues. This advanced technique enhances spatial resolution for immune profiling in various disease contexts.
Area of Science:
- Biomedical Imaging
- Molecular Pathology
- Immunohistochemistry
Background:
- Tissues exhibit complex cellular heterogeneity, requiring advanced imaging to resolve immune cell interactions.
- Conventional methods like single-cell suspension lack spatial context crucial for understanding pathology.
- High-dimensional imaging mass cytometry (IMC) allows simultaneous detection of up to 40 protein markers at subcellular resolution.
Purpose of the Study:
- To optimize a staining protocol for imaging mass cytometry (IMC).
- To integrate RNAscope technology with IMC for combined protein and RNA detection.
- To expand IMC utility for investigating low-abundance or noncoding RNA targets within tissue microenvironments.
Main Methods:
- Development and optimization of a novel staining protocol for IMC.
- Integration of RNAscope in situ hybridization with IMC.
- Validation of the protocol for concurrent protein and single-molecule RNA detection in tissue samples.
Main Results:
- Successful integration of RNAscope with IMC, enabling multiplexed protein and RNA analysis.
- Demonstration of enhanced capability for detecting low-abundance and noncoding RNA targets.
- Preservation of subcellular resolution for both protein and RNA markers.
Conclusions:
- The optimized protocol significantly expands the capabilities of IMC for comprehensive immune profiling.
- This method provides crucial spatial context for studying host-pathogen interactions, tumor microenvironments, and inflammatory diseases.
- The combined approach offers a powerful tool for advancing our understanding of complex biological systems within their native tissue context.
Abstract:
The complexities and cellular heterogeneity associated with tissues necessitate the concurrent detection of markers beyond the limitations of conventional imaging approaches in order to spatially resolve the relationships between immune cell populations and their environments. This is a necessary complement to single-cell suspension-based methods to inform a better understanding of the events that may underlie pathological conditions. Imaging mass cytometry is a high-dimensional imaging modality that allows for the concurrent detection of up to 40 protein markers of interest across tissues at subcellular resolution. Here, we present an optimized staining protocol for imaging mass cytometry with modifications that integrate RNAscope. This unique addition enables combined protein and single-molecule RNA detection, thereby expanding the utility of imaging mass cytometry to researchers investigating low abundance or noncoding targets. In general, the procedure described is broadly applicable for comprehensive immune profiling of host-pathogen interactions, tumor microenvironments and inflammatory conditions, all within the tissue contexture.
